Hfq is an RNA binding protein that plays multiple roles in the post-transcriptional regulation of gene expression in bacteria. In cases where the gene in question is a transcriptional regulator, the presence or absence of Hfq can also influence the expression patterns of the subsets of genes controlled by this regulator. Previous studies in our laboratory have shown that Hfq is required for the wild-type virulence of Brucella abortus 2308 in the mouse model. To date, we have identified >40 genes that require Hfq for their wild-type expression in this strain, and seven of these (sodC, ahpCD, virB, bvrRS, omp25, cydAB and znuA) are necessary for virulence in mice! RT-PCR analysis indicates that sodC, ahpCD, virB.and cydAB display both Hfq-dependent and Hfq-independent patterns of transcription in 6. abortus 2308, while bvrRS transcription appears to be Hfq-dependent under all experimental conditions that have been examined. These experimental findings suggest that a transcriptional activator or alternativeCTfactor serves as an intermediate regulatory link between Hfq and wild-type expression of sodC, bvrRS, virB and cydAB in B. abortus 2308. Our phenotypic analyses of B. abortus alternative factor a null mutants, however, suggest that an alternative a factor is not playing this role. Consequently, in order to gain a better understanding of the regulatory link between Hfq and sodC, ahpCD, virB, bvrRS, and cydAB in B. abortus 2308, the specific aims of the studies outlined in this application are a) to test the hypothesis that a transcriptional activator is responsible for Hfq-dependent expression of sodC, ahpCD, bvrRS, virB and cydAB in this strain;b) to identify this Hfq-dependent transcriptional regulator (HtcR);and c) to determine the extent to which the HtcR- dependent promoter regions of sodC, ahpCD, bvrRS, virB and cydAB are required for the virulence of B. abortus 2308 in mice. Information derived from the proposed studies will provide insight into the mechanisms by which the brucellae regulate their gene expression during residence in their mammalian hosts, and such information should be useful for the design of novel vaccine candidates and improved chemotherapeutic approaches. These studies may also identify a novel mechanism for regulating stationary phase gene expression in prokaryotes. The Brucella spp. do not possess a homolog of the alternative a factor RpoS which performs this function in many Gram-negative bacteria, but Hfq appears to be required for the efficient stationary phase expression of socfC, ahpCD, virB and cydAB in B. abortus 2308.